American Society of Hematology

Fig. 2.

$Fig. 2. Activation of Cdc42 and Rac1 in platelets. / (A) Specific interaction of Cdc42-GTP or Rac1-GTP with PBD. Platelet lysates were incubated with 100 μM GTPγS or GDP and clarified before precipitation on PBD-GST beads. Supernatants (S) were separated from bead pellets (P) by centrifugation and analyzed by SDS-PAGE and specific immunoblotting (IB). (B) Time course of Cdc42 or Rac1 activation in stimulated platelets. After stimulation with 0.5 UI/mL THR (⋄), 10 μM TRAP (■), 250 pmol/L CVX (○), or 2 μg/mL monoclonal IV.3 antibody followed by 30 μg/mL antimouse IgG F(ab′)2 (▵), platelets were lysed in 1% NP-40 buffer. Pull-down of GTP-bound Cdc42 or Rac1 on PBD-GST beads was performed. (Upper panel) Representative anti-Cdc42 or anti-Rac1 immunoblots. (Lower panel) Quantification of Cdc42-GTP or Rac1-GTP expressed as mean ± SD of 3 separated experiments. (C) Expression of Cdc42 in cytoskeleton pellets (P) and NP-40–soluble supernatants (S) of CVX- or IV.3-stimulated platelets. NP-40–insoluble and –soluble fractions were separated by centrifugation (10 000g for 20 minutes at 4°C), and samples corresponding to 15 × 106 platelets were analyzed for Cdc42 expression by immunoblotting. Results are representative of 3 separate experiments.$

Activation of Cdc42 and Rac1 in platelets.

(A) Specific interaction of Cdc42-GTP or Rac1-GTP with PBD. Platelet lysates were incubated with 100 μM GTPγS or GDP and clarified before precipitation on PBD-GST beads. Supernatants (S) were separated from bead pellets (P) by centrifugation and analyzed by SDS-PAGE and specific immunoblotting (IB). (B) Time course of Cdc42 or Rac1 activation in stimulated platelets. After stimulation with 0.5 UI/mL THR (⋄), 10 μM TRAP (■), 250 pmol/L CVX (○), or 2 μg/mL monoclonal IV.3 antibody followed by 30 μg/mL antimouse IgG F(ab′)₂ (▵), platelets were lysed in 1% NP-40 buffer. Pull-down of GTP-bound Cdc42 or Rac1 on PBD-GST beads was performed. (Upper panel) Representative anti-Cdc42 or anti-Rac1 immunoblots. (Lower panel) Quantification of Cdc42-GTP or Rac1-GTP expressed as mean ± SD of 3 separated experiments. (C) Expression of Cdc42 in cytoskeleton pellets (P) and NP-40–soluble supernatants (S) of CVX- or IV.3-stimulated platelets. NP-40–insoluble and –soluble fractions were separated by centrifugation (10 000g for 20 minutes at 4°C), and samples corresponding to 15 × 10⁶ platelets were analyzed for Cdc42 expression by immunoblotting. Results are representative of 3 separate experiments.

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